The present invention relates to technology for restoring soil and preventing contamination of ground water by decomposing organic compounds by decomposing microorganisms in, for example, soil that has been contaminated by organic compounds such as agricultural chemicals. In particular, the present invention relates to technology for rapidly and efficiently accumulating and isolating only those decomposing microorganisms of a species that is capable of decomposing poorly degradable organic contaminants contained in soil, and to technology for restoring contaminated soil and preventing contamination of ground water by applying a retentive carrier for decomposing microorganisms that has accumulated those isolated decomposing microorganisms directly to contaminated soil.
The restoration of soil contaminated by organic compounds such as agricultural chemicals by decomposing microorganisms along with the prevention of ground water contamination caused by those organic compounds has conventionally been considered to be a useful technology. For example, if decomposing microorganisms that decompose or detoxify organic compounds such as agricultural chemicals are present among microorganisms that thrive in soil, and the capabilities of these decomposing microorganisms is utilized, contaminants such as agricultural chemicals can be removed from soil.
However, in the natural state, due to the low density of decomposing microorganisms having the ability to decompose specific organic compounds, it has not been possible to effectively prevent the retention or diffusion of contaminants in the environment.
Consequently, if it were possible to accumulate only decomposing microorganisms able to decompose specific organic compounds at a higher density and in a state in which they possess decomposing activity, it would be possible to effectively realize restoration of contaminated soil and prevention of ground water contamination.
However, in the case of methods for culturing decomposing microorganisms carried out in the prior art, such as a method in which only a specific decomposing microorganism is inoculated into and grown in a liquid medium or solid medium that only uses a specific organic compound for its carbon source and nitrogen source, although only a specific decomposing microorganism can be cultured at high density, even if only that cultured decomposing microorganism is applied directly to an environment such as contaminated soil, that cultured decomposing microorganism hardly ever demonstrates its decomposing activity in a stable manner for a long period of time.
This is because, in the case of directly applying only a specific decomposing microorganism to contaminated soil, as a result of being subjected to the physiochemical properties of the contaminated soil, being preyed on by the large numbers of Protozoa and other organisms that thrive in the contaminated soil, or being unable to acquire a niche due to competition with other microorganisms, that specific decomposing microorganism is destroyed. In addition, this is also because, if the target organic compound to serve as the nutrient source (carbon source and nitrogen source) of a specific decomposing microorganism is adsorbed into a soil solid phase and is in a state that cannot be supplied to the specific decomposing microorganism, namely if it is in a state of low bioavailability, the decomposing activity of that specific decomposing microorganism cannot be demonstrated and maintained.
In consideration of these circumstances, the object of the present invention is to provide a technology for rapidly and efficiently accumulating a decomposing microorganism of a species that is capable of decomposing a specific organic compound in a state in which that decomposing activity is demonstrated in a stable manner for a long period of time, and for restoring contaminated soil and preventing contamination of ground water by using a decomposing microorganism retentive carrier that has accumulated that decomposing microorganism to decompose organic compounds present in contaminated soil.
The present invention is based on new findings obtained from research on an improved soil perfusion method previously proposed by the inventors of the present invention (Japanese Patent Application No. Hei 9-30176). The improved soil perfusion method proposed by the inventors of the present invention refers to using a porous material, which together with having a function that adsorbs a specific organic compound, also has a countless number of pores in a state that facilitates habitation by decomposing microorganisms having the ability to decompose that organic compound, to form an accumulation soil layer composed of this porous material and soil in which said decomposing microorganisms thrive, followed by continuously perfusing an inorganic salt medium having only the target organic compound for its carbon source and nitrogen source through this accumulation soil layer, thereby rapidly accumulating decomposing microorganisms in said porous material.
As a result, the inventors of the present invention considerably improved the accumulation and isolation rates of decomposing microorganisms to an extent that was unable to be realized with the soil perfusion method of the prior art. In other words, in this improved soil perfusion method, the porous material efficiently adsorbs carbon and nitrogen sources contained in the inorganic salt medium, namely the organic compound that is contaminating the soil, and due to the favorable conditions imparted by its pores, decomposing microorganisms grow and function actively by using the adsorbed organic compound as a nutrient source (carbon source and nitrogen source). As a result of effectively combining these factors, organic compounds that have been efficiently adsorbed into the pores are efficiently assimilated and decomposed by decomposing microorganisms within the pores, thereby realizing their rapid accumulation.
Decomposing microorganisms that accumulate in the porous material according to this improved soil perfusion method are in a state in which decomposing microorganisms of a species that has the ability to decompose a specific organic compound are selectively accumulated. However, the decomposing microorganisms that have accumulated in the porous material are actually in the state of a mixture with several species of bacteria not directly involved in decomposition.
Therefore, the inventors of the present invention attempted to apply the above improved soil perfusion method in order to accumulate only decomposing microorganisms of a species that is capable of decomposing a specific organic compound in a purified state. More specifically, according to the above improved perfusion method, a porous material that had accumulated a specific decomposing microorganism was first used as the inoculation source to inoculate a fresh porous material (in which decomposing microorganisms had not yet been accumulated) in a state of being mixed with several species of bacteria not directly involved in decomposition. By then perfusing an inorganic salt medium having for its carbon source and nitrogen source only the organic compound that serves as the nutrient source of that specific decomposing microorganism, only the specific decomposing microorganism was purified and accumulated in the fresh porous material.
Although it is possible that the decomposing microorganism accumulated in the porous material following the above purification is a single species of decomposing microorganism, since normally two to three species of decomposing microorganisms are present, in order to accumulate only those decomposing microorganisms that decompose a specific organic compound, the porous material in which decomposing microorganisms are accumulated following purification was crushed, mixed dilutions of the crushed porous material were prepared using phosphate buffer as diluent, those mixed dilutions were plated onto inorganic salt agar media that uses only the specific organic compound for its carbon source and nitrogen source, and microorganisms were picked from those portions of high decomposing activity formed on the inorganic salt agar medium (clear zones) to isolate only specific decomposing microorganisms.
Moreover, only specific decomposing microorganisms picked and isolated from the clear zone formed on inorganic salt agar media were used as an inoculation source to inoculate a fresh porous material, and an inorganic salt medium having only a specific organic compound for its carbon source and nitrogen source was perfused through that porous material. As a result, it was ultimately determined that only decomposing microorganisms of a species that is able to decompose a specific organic compound were able to be accumulated in the porous material at a high density in an extremely purified state. Decomposing microorganisms accumulated at high density and in an extremely purified state in a porous material in this manner were determined to decompose an organic compound adsorbed to the porous material in a stable manner.
According to the research results of the inventors of the present invention as described above, it was determined that the technology described below enables accumulation of decomposing microorganisms. To begin with, as is described in claim 1, only decomposing microorganisms of a species able to decompose a specific organic compound are inoculated into a porous material, which together with being able to adsorb said organic compound, has a countless number of pores that facilitate habitation by said decomposing microorganisms, an accumulation layer is formed from the inoculated porous material, and the inorganic salt medium, having only the above organic compound for its carbon source and nitrogen source, is perfused through this accumulation layer.
As a result, only a decomposing microorganism of a species that is able to decompose a specific organic compound can be accumulated at high density in a porous material both rapidly and efficiently. Decomposing microorganisms accumulated in a porous material in this manner are able to demonstrate and maintain stable decomposing activity on the organic compound adsorbed to the porous material.
In both that previously described and that described below, decomposing microorganisms of a species that are able to decompose a specific organic compound refer to that which employs the concept that includes not only the case of a mixture of a plurality of species of decomposing microorganisms, but also the case of only a single species of decomposing microorganism.
It is necessary to identify only those decomposing microorganisms in order to inoculate only a species of decomposing microorganism able to decompose a specific organic compound into a porous material. In this case, as is described in claim 2, it is preferable to inoculate a group of bacteria containing a species of decomposing microorganisms that is able to decompose a specific organic compound onto an inorganic salt agar medium that only has said organic compound for its carbon source and nitrogen source, pick microorganisms from a portion of high decomposing activity that have formed on the inorganic salt agar medium (clear zone), and inoculate only the isolated decomposing microorganism.
When decomposing microorganisms picked and isolated from a portion of high decomposing activity formed on the inorganic salt agar medium, namely the portion at which decomposing microorganisms able to decompose a specific organic compound are thriving, such as decomposing microorganisms obtained by cutting out a portion of a clear zone in a state in which the decomposing microorganisms are accompanied by inorganic salt agar medium, are inoculated into a porous material, only those decomposing microorganisms of a species that is able to decompose a specific organic compound can be rapidly and efficiently accumulated. This is because, although there is a method in which decomposing microorganisms are grown and inoculated from so-called liquid medium, according to current research results of the inventors of the present invention, an inoculation method consisting of cutting out clear zones formed on inorganic salt agar medium for each agar medium has been confirmed to allow rapid and efficient accumulation in the present invention.
Moreover, in the case of accumulating only a decomposing microorganism of a species that is able to decompose an organic contaminant in which a specific organic compound can be contained in contaminated soil by selecting that decomposing microorganism from contaminated soil, as described in claim 3, it is preferable to accumulate only said decomposing microorganism in a porous material by removing an accumulated porous material that has accumulated decomposing microorganisms obtained according to the above improved soil perfusion method from an accumulation soil layer, perfusing the inorganic salt medium having only said organic contaminant for its carbon source and nitrogen source through the accumulation soil layer, removing the accumulated porous material from the accumulation soil layer, performing washing treatment that removes soil adhering to the removed accumulated porous material, using the washed accumulated porous material as an inoculation source to inoculate an accumulation layer formed only from a fresh porous material, using a group of bacteria accumulated by purifying in the fresh porous material by perfusing an inorganic salt medium having only said organic contaminant for its carbon source and nitrogen source through the accumulation layer, and using the inorganic salt agar medium described in claim 2 to isolate the decomposing microorganisms.
When this is done, only decomposing microorganisms having high decomposing activity for a specific organic contaminant can be selected from bacteria thriving in contaminated soil, making it possible to rapidly and efficiently accumulate those decomposing microorganisms in a porous material at high density. This is effective in the case of decomposing an organic chlorine-based compound such as an agricultural chemical known to be a soil contaminant, and according to research conducted by the inventors of the present invention, has been demonstrated by using soil containing the poorly degradable organic chlorine-based compounds of simazine (CAT) and quintozene (PCNB).
Porous materials in which only decomposing microorganisms of a species able to decompose a specific organic compound are accumulated by the accumulation method for isolated decomposing microorganisms as claimed in the present invention offer the following advantages as decomposing microorganism retentive carriers. These advantages consist of these porous materials allow decomposing microorganisms to be handled extremely easily and being able to be easily applied to soil. In addition, in the case of using charcoal or other ligneous material as a porous material, there are no detrimental effects on the soil when applied directly to the soil, and these materials have properties that allow contaminants in the form of organic compounds to be adsorbed efficiently.
Moreover, as is described in claim 6, by using a porous material that has accumulated only a specific decomposing microorganism obtained by the aggregation method for isolated decomposing microorganisms as claimed in the present invention as a decomposing microorganism retentive carrier, and burying that retentive carrier in contaminated soil containing the target organic compound, the organic compound in the contaminated soil can be decomposed, making it possible to restore contaminated soil and prevent ground water contamination.
In the case of burying the decomposing microorganism retentive carrier as claimed in the present invention in contaminated soil, it is preferable to thoroughly mix with the contaminated soil. This is because, when buried after thoroughly mixing with the contaminated soil in this manner, there is greater contact between the contaminated soil and decomposing microorganism retentive carrier, resulting in the target organic compound being uniformly adsorbed onto the porous material. In addition, it is also preferable to perfuse an inorganic salt medium having only the target organic compound for its carbon source and nitrogen source through the contaminated soil in which the decomposing microorganism retentive carrier is buried. This is because, when this is done, recovery treatment of the contaminated soil can be performed rapidly and efficiently. Since organic compounds contained in contaminated soil flow through the soil due to penetration of sprayed water and rain water, a specific organic compound contained in the contaminated soil can be decomposed simply by burying the decomposing microorganism retentive carrier as claimed in the present invention in the contaminated soil without having to perfuse with the inorganic salt medium as described above.
Decomposing a specific organic compound in contaminated soil by a specific decomposing microorganism accumulated in a porous material even if the decomposing microorganism retentive carrier as claimed in the present invention is buried in contaminated soil in this manner is thought to be possible for the reasons indicated below.
To begin with, since decomposing microorganisms thrive inside pores of a porous material in the decomposing microorganism retentive carrier as claimed in the present invention, the porous material fulfills the role of a so-called shelter, and is presumed to protect the accumulated specific decomposing microorganisms from the physicochemical properties of the contaminated soil, from being preyed on by the large numbers of Protozoa and other organisms that thrive in the contaminated soil, or from competition with other microorganisms over acquiring a niche and so forth. Since the porous material efficiently adsorbs organic compounds in contaminated soil, by providing a nutrient source (carbon source and nitrogen source) of a specific decomposing microorganism in the pores, a state of high bioavailability results, which is presumed to enable decomposing activity to be demonstrated and maintained in a stable manner.
Thus, according to the present invention, restoration treatment of contaminated soil can be performed rapidly and efficiently, and even if an organic compound such as an agricultural chemical is again sprayed on the contaminated soil, since the decomposing microorganism retentive carrier is buried in the contaminated soil, the sprayed, for instance, organic compound is decomposed, thereby preventing its retention and diffusion into the soil. Entry of organic compounds present in the soil into ground water is also avoided, thereby making it possible to prevent contamination of ground water. Various applications can be considered based on these characteristics of the present invention. Examples of these include, with respect to aqueous systems, domestic wastewater lines, agricultural wastewater lines in areas with rice paddies and the wastewater lines of golf courses, with respect to soil systems, top and bottom soil containing environmental contaminants, bottom soil of the greens of golf courses, bottom soil of industrial waste processing sites and bottom soil of organic waste storage sites at factories and so forth, and with respect to combined aqueous and soil systems, soil at coastlines contaminated by runoff oil. Direct application of the decomposing microorganism retentive carrier as claimed in the present invention to these applications results in effective environmental protection.
The phenomenon in the present invention described above is an extremely rare example throughout the world in technical fields utilizing microorganisms. Consequently, the present invention can be said to be an extremely beneficial technology especially today when there is a dire need for preventing environmental contamination expanding on a global scale as quickly as possible for the purpose of restoring the contaminated environment (bioremediation).